This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production and isolation of such polynucleotides and polypeptides. More particularly, the polynucleotides and polypeptides of the present invention have been identified as being variant porcine stem cell factors, and in particular membrane-bound stem cell factors, and still more particularly as being involved in stem cell, useful for supporting proliferation and growth of porcine bone marrow cells.
The present invention provides in one aspect a novel polypeptide which has been characterized as an active form of porcine stem cell factor cDNA gene sequence(PSCF) that omits exon 6. Preferably, the PSCF is a splice variant wherein exon 6 is omitted. More preferably, exon 6 is removed from the native full-length porcine stem cell factor and is omitted entirely or is replaced with a polynucleotide that encodes one or more amino acids.
In a preferred aspect the invention provides a PSCF encoded by a polynucleotide sequence corresponding to the full-length native porcine stem cell factor cDNA, but in which: (1) the first 70 nucleotides are removed, (2) exon 6 is excised (nucleotides 591 to 654) from the full polynucleotide sequence, (3) the excised exon 6 segment is replaced by a three-nucleotide segment encoding the amino acid xe2x80x9cGlyxe2x80x9d, and (4) the fifteen-nucleotide C-terminal tail (nucleotides 938-952) is removed and replaced by the six-nucleotide segment 5xe2x80x2-TCTAGA-3xe2x80x2 (SEQ ID NO: 11).
In accordance with another aspect of the present invention, there are provided novel PSCF polypeptides, as well as active fragments, analogs and derivatives thereof. In a preferred aspect the present invention provides novel membrane-bound PSCF polypeptides, wherein the polypeptide segment encoded by exon 6 is omitted or replaced by an inactive polypeptide segment. In another preferred aspect the present invention provides such novel PSCF polypeptides which are soluble PSCF polypeptides.
In accordance with another aspect of the present invention, there are provided isolated nucleic acid molecules encoding the polypeptides of the present invention including mRNAs, cDNAs, genomic DNAs as well as active analogs and fragments of such polypeptides.
In accordance with yet a further aspect of the present invention, there is provided a process for producing such polypeptides by recombinant techniques comprising culturing recombinant prokaryotic and/or eukaryotic host cells, containing a nucleic acid sequence of the present invention, under conditions promoting expression of said polypeptides.
In accordance with a further aspect of the invention, the polypeptide of the invention may be anchored to a cell""s surface, and the modified cell, as a feeder cell for culturing porcine cells.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides for analyzing potential agonists to the polypeptides, Another process utilizes the polynucleotides to assay for compounds which bind said polynucleotides and would thus block expression of any products from said polynucleotides.
In accordance with yet a further aspect of the present invention, there are also provided nucleic acid probes comprising nucleic acid molecules of sufficient length to specifically hybridize to a nucleic acid sequence of the present invention.
In accordance with yet a further aspect of the present invention, there is provided a process for utilizing such polypeptides, or polynucleotides encoding such polypeptides, for purposes related to scientific research for example, to generate probes for identifying similar sequences which might encode similar polypeptides from other organisms by using certain regions, i.e., conserved sequence regions, of the nucleotide sequence.
In accordance with an aspect of the present invention, there are provided isolated nucleic acids (polynucleotides) which encode PSCF polypeptides, in which polynucleotides exon 6 has been removed, deactivated or replaced by an inactive portion (SEQ ID NO:8) as shown in FIG. 4, as well as said encoded mature PSCF polypeptide as shown in FIG. 4 (SEQ ID NO:9).
These and other aspects of the present invention should be apparent to those skilled in the art from the teachings herein.
In order to facilitate understanding of the following description and examples which follow certain frequently occurring methods and/or terms will be described.
The term xe2x80x9cgenexe2x80x9d means the segment of DNA involved in producing a polypeptide chain; it includes regions preceding and following the coding region (leader and trailer) as well as intervening non-coding sequences (introns) between individual coding segments (exons).
A coding sequence is xe2x80x9coperably linked toxe2x80x9d another coding sequence when RNA polymerase will transcribe the two coding sequences into a single mRNA, which is then translated into a single polypeptide having amino acids derived from both coding sequences. The coding sequences need not be contiguous to one another so long as the expressed sequences ultimately process to produce the desired polypeptide.
xe2x80x9cRecombinantxe2x80x9d polypeptides refer to polypeptides produced by recombinant DNA techniques; i. e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide. xe2x80x9cSyntheticxe2x80x9d polypeptides are those prepared by chemical synthesis.
A DNA xe2x80x9ccoding sequence ofxe2x80x9d or axe2x80x9cnucleotide sequence encodingxe2x80x9d a particular polypeptide, is a DNA sequence which is transcribed and translated into a polypeptide when placed under the control of appropriate regulatory sequences.
xe2x80x9cPlasmidsxe2x80x9d are designated by a lower case xe2x80x9cpxe2x80x9d preceded and/or followed by capital letters and/or numbers. The starting plasmids herein are either commercially available, publicly available on an unrestricted basis, or can be constructed from available plasmids in accord with published procedures. In addition, equivalent plasmids to those described are known in the art and will be apparent to the ordinarily skilled artisan.
xe2x80x9cDigestionxe2x80x9d of DNA refers to catalytic cleavage of the DNA with a restriction enzyme that acts only at certain sequences in the DNA. The various restriction enzymes used herein are commercially available and their reaction conditions, cofactors and other requirements were used as would be known to the ordinarily skilled artisan. For analytical purposes, typically 1 xcexcg of plasmid or DNA fragment is used with about 2 units of enzyme in about 20 xcexcl of buffer solution. For the purpose of isolating DNA fragments for plasmid construction, typically 5 to 50 xcexcg of DNA are digested with 20 to 250 units of enzyme in a larger volume. Appropriate buffers and substrate amounts for particular restriction enzymes are specified by the manufacturer. Incubation times of about 1 hour at 37xc2x0 C. are ordinarily used, but may vary in accordance with the supplier""s instructions. After digestion the reaction is electrophoresed directly on a polyacrylamide gel or an agarose gel to isolate the desired fragment.
xe2x80x9cOligonucleotidesxe2x80x9d refers to either a single stranded polydeoxynucleotide or two complementary polydeoxynucleotide strands which may be chemically synthesized. Such synthetic oligonucleotides have no 5xe2x80x2 phosphate and thus will not ligate to another oligonucleotide without adding a phosphate group with an ATP in the presence of a kinase. A synthetic oligonucleotide will ligate to a fragment that has not been dephosphorylated.
xe2x80x9cLigationxe2x80x9d refers to the process of forming phosphodiester bonds between two double stranded nucleic acid fragments (J. Sambrook et al., 1989, in Molecular Cloning. A Laboratory Manual, Cold Spring Harbor Laboratory, NY). Unless otherwise provided, ligation may be accomplished using known buffers and conditions with 10 units of T4 DNA ligase (xe2x80x9cligasexe2x80x9d) per 0.5 xcexcg of approximately equimolar amounts of the DNA fragments to be ligated.
Unless otherwise stated, transformation was performed as described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, 1989.
xe2x80x9cPorcine Stem Cell Factorxe2x80x9d (PSCF) or xe2x80x9cporcine steel factorxe2x80x9d are terms that are used herein interchangeably. Also the corresponding mouse factors are discussed as being xe2x80x9cmouse stem factorxe2x80x9d (MSF) or xe2x80x9cmouse steel factorxe2x80x9d. PSCF is also called factor, porcine mast cell growth factor and porcine c-kit ligand in the art. Each of the native steel factors (SFs) has a transmembrane polypeptide with a cytoplasmic domain and an extracellular domain. Soluble PSCF or MSF refer to a fragment cleaved from the extracellular domain at a specific proteolytic cleavage site (e.g. for soluble PSCF, amino acids 1-160. Membrane associated SF refers to both normal SF before it has been cleaved or the SF which has been altered so that proteolytic cleavage cannot take place. The PSCF may be either a soluble form as described in U.S. Pat. No. 5,589,582, or its equivalents, or may be a membrane-bound form, preferably bound on to the surface of a cell.
xe2x80x9cFeeder cellsxe2x80x9d are cells which produce membrane-bound and/or soluble PSCF, preferably a fibroblast cell that is transformed to produce the PSCF, more preferably are transformed murine fibroblast cells, and even more preferably are murine fibroblast cells known as the STO cell line that have been transformed. Particularly, preferred feeder cells have the polypeptide according to the invention anchored to the surface of the cell. Such feeder cell lines may be referred to above and hereinafter as xe2x80x9cSTO5xe2x80x9d, xe2x80x9cSTO8xe2x80x9d, xe2x80x9cSTO12xe2x80x9d or xe2x80x9cSTO18xe2x80x9d cells. Feeder cells which produce murine stem cell factor MSCF may be referred to as STO cells (the STO cell line is a thioguanine/oubain resistant sub-line of SIM mouse fibroblasts, Virology 50:339 (1972); STO cells are described in U.S. Pat. No. 5,453,357). The full-length amino acid sequence for PSCF (SEQ ID NO:2) based on native cDNA (SEQ ID NO:1 and FIG. 1 attached hereto) is reported in Biology of Reproduction 50: 95-102 (1994), and active forms can be produced by transfecting cells with active fragments of the cDNA sequence which may have the polynucleotides encoding the leader sequence amino acids (xe2x88x9225 to xe2x88x921) removed. Soluble forms preferably omit the polynucleotide transmembrane portion. Additionally, an active form of PSCF (as in the present invention) can be produced and utilized to transfect STO cells by removing exon 6 from the full length cDNA and substituting a polynucleotide segment that encodes one or more amino acids. Active forms of the PSCF of the present invention may also omit the C-terminal polypeptide segment corresponding to native polypeptide beginning with amino acid 217 (see, FIG. 1 or SEQ ID NO:2 for such C-terminal segment).